The long term goal of this research is to understand a signaling pathway that regulates programs of gene expression by controlling the subcellular localization of regulatory proteins. In the fruit fly Drosophila, signal transduction by the cell surface receptor Toll promotes nuclear translocation of transcription factors governing dorsoventral axis formation in the embryo and innate immunity in the adult. Pathway function in innate immune responses has been widely conserved, with homologous pathways inducing antimicrobial defenses in both plants and mammals. In flies, the targets of Toll signaling are Dorsal and Dif, a pair of NF-KappaB related transcription factors, and the inhibitory protein Cactus. Signal transduction from Toll to Cactus and Dorsal/Dif requires two adaptor proteins, MyD88 and Tube, as well as a protein kinase, Pelle. The fact that these pathway components have been characterized at both the genetic and molecular level and can be assayed in embryo, whole fly, and cell culture systems makes this pathway particularly amenable to experimental investigation. It is now possible, therefore, to address fundamental questions about the mechanism for signal transduction. The focus of the proposed research will be to move from a static picture of a series of protein interactions to a dynamic understanding of signal transduction in the context of overall pathway architecture. We will develop reagents for the rapid and synchronous initiation of signaling, then exploit these tools to probe how pathway components oligomerize, associate and dissociate, and are modified, relocalized and degraded, to effect signal transduction. In carrying out these studies, we will exploit inducible dimerization systems for triggering pathway activation, RNAi technology for large-scale reverse genetic screens, site directed modifications of the Pelle protein kinase to identify direct targets of catalysis, and fluorescence resonance energy transfer (FRET) to monitor the assembly of protein complexes. At the same time, we will carry out studies of cis-regulatory sequences in target genes, focusing on integration of Toll pathway signals into the overall innate immune response. Given the conserved nature of the signaling pathway, the results of the proposed research should be of substantial interest with regard to signal transduction and innate immunity in a broad range of organisms.